Characterization of Rimonabant effects on G protein activity

G protein-coupled receptor (GPCRs) is the largest class of cell-surface receptors, and represents today the target of 40% of the drugs in the pharmaceutical market. In the absence of agonists, many GPCRs have found to exhibit spontaneous activity, which can be blocked by ligands that are referred to as inverse agonists (Milligan, 2003). Cannabinoid CB1 receptor is one of the most abundant GPCR in the central nervous system, and is coupled to Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinase (MAPK), inhibit voltage gated Ca2+ channels and activate inwardly rectifying K+ channels (Howlett et al., 2000; Pertwee, 2010). The first selective and potent CB1 antagonist Rimonabant (also known SR141716A, SR) at high micromolar concentrations behaves as an inverse agonist, i.e. decreases [35S]GTPγS binding in rodent and human cerebral cortex and in Chinese hamster ovary (CHO) cells transfected with CB1 receptors (Rinaldi-Carmona, 1994). However, in vitro and in vivo studies performed using CB1 knockout (KO) and CHO cells not expressing CB1 receptors suggest that inverse agonist activity of SR is CB1 receptor independent (Pertwee, 2005). Several hypotheses have been postulated to explain the inverse agonism of SR, including its action on different receptors (i.e GPCR mainly coupled to Gi/o proteins) and/or its negative modulation of the constitutive activity of CB1 receptors. Alternatively, SR might explicate these “inverse agonist effect” in a manner receptor-independent acting directly on G protein level. The present study aimed to determine whether the CB1 receptor-independent effects of SR are mediated via GPCRs, in particular GABAB and dopamine D2 receptors, that share the same Gαi/o signaling pathways, or if SR acts directly on G protein subunits. For this purpose we investigated the molecular mechanisms of SR on G protein activity in native and recombinant systems by using different experimental approaches (i.e., GTPγS binding, bioluminescence Resonance Energy Transfer (BRET), electrophysiological recordings). In particular, we first evaluated the effects of SR on basal and agonist-stimulated [35S]GTPγS binding in systems containing CB1, GABAB and D2 receptor populations (i.e., rat membrane homogenates and CHO stable transfected with GABAB or D2 receptors), and in systems lacking CB1 and GABAB receptors (i.e., CB1- and GABAB-KO mice). Then, using BRET approach we monitored dissociation between Gαo and Gβγ subunits and their conformational rearrangements before and after GABAB receptors activation. In addition, using the same assay we studied the molecular interaction between D2 receptor and Gαi1 protein subunits (Gαi1-60, Gαi1-91 and Gαi1-121). Next, we evaluated the effects of SR on adenylate cyclase activity, using BRET with the CAMYEL sensor, a recent technique developed to detect the level of cAMP in living cells. Specifically, the inhibitory effect of SR on Gi and Gs protein pathways measuring BRET signal in cells transfected with CAMYEL and GABAB, D2 or D1 receptors was investigated. Finally, whole cell voltage clamp recordings from midbrain dopamine neurons in acute rat brain slices ex vivo were performed to evaluate the effects of SR on baclofen and quinpirole-induced outward K+ current both in wild-type (WT) and CB1-KO mice. In addition, in order to demonstrate that SR induced the inhibition of GIRK channel activity by acting directly on G protein, we use a GPCR-free experimental setup (i.e. whole cell patch clamp experiments were performed in CHO cells transfected with GIRK1/2). The main finding of this study is that SR, at micromolar concentrations, prevented GPCR-G protein signaling through a direct interaction with the G proteins mainly with the subunits αi/o.